Analysis of the haemolysin secretion system by PhoA-HlyA fusion proteins.
We studied the efficiency of the pHly152-derived haemolysin transport system using PhoA-HlyA fusion proteins and different constructs which provide HlyB/HlyD in trans. The optimal C-terminal HlyA signal consists of the last 60 amino acids. Longer stretches of HlyA do not improve the transport efficiency of PhoA-HlyA fusion proteins. The introduction of deletions and/or replacements in the 60 amino acid HlyA signal domain revealed at least three functional regions with different degrees of specificity. Amino acids 1-21 (numbered from the N-terminal part of the 60 amino acid HlyA signal), termed region I, could be replaced by a Pro-containing peptide. The other two regions II and III (amino acids 22-40 and 41-60, respectively) seem to interact directly with the HlyB/HlyD translocator since a PhoA fusion protein which contains either of the two regions was still secreted in a HlyB/HlyD-dependent mode, albeit at low efficiency. An efficient trans-complementing HlyB/HlyD system was only obtained from the pHLy152-encoded hly determinant when the regulatory hlyR element was provided in cis. Secretion of the PhoA-HlyA fusion protein did not interfere with the secretion of HlyA even when the fusion protein was induced to a high level. This suggests that the capacity of the HlyB/HlyD translocation system is high and not normally saturated by its natural HlyA substrate.
Hess
J
J
Institut für Genetik und Mikrobiologie, Universität Würzburg, Federal Republic of Germany.
Gentschev
I
I
Goebel
W
W
Jarchau
T
T
D000595:Amino Acid Sequence; D001425:Bacterial Outer Membrane Proteins / Q000235:genetics; D001426:Bacterial Proteins / Q000235:genetics* / Q000378:metabolism; D001483:Base Sequence; D002352:Carrier Proteins / Q000235:genetics; D002872:Chromosome Deletion; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000235:genetics* / Q000378:metabolism; D029968:Escherichia coli Proteins; D006460:Hemolysin Proteins / Q000235:genetics*; D008969:Molecular Sequence Data; D015345:Oligonucleotide Probes; D010957:Plasmids; D021382:Protein Sorting Signals / Q000235:genetics / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D015183:Restriction Mapping
D001425:Bacterial Outer Membrane Proteins; D001426:Bacterial Proteins; D002352:Carrier Proteins; D029968:Escherichia coli Proteins; D006460:Hemolysin Proteins; C036507:Hlya protein, E coli; C050837:Hlyb protein, Bacteria; D015345:Oligonucleotide Probes; D021382:Protein Sorting Signals; D011993:Recombinant Fusion Proteins